The invention relates to screening procedures which identify compounds for inhibiting infection or disease in a eukaryotic host organism, or which induce or stimulate a host""s pathogenic defense mechanisms. The invention also relates to the use of such compounds as anti-pathogens. In addition, the invention relates to procedures which identify pathogenic virulence factors.
Microbial pathogens such as bacteria, protozoa, fungi, nematodes, and viruses include a large and diverse group of organisms capable of infecting animals and plants. Initiation of an infection occurs when the infecting organism is pathogenic, and the host is susceptible to pathogenic invasion. After establishing contact with susceptible cells or tissues of the host, the pathogen acquires nutrients from its host, facilitating its own survival. During the infection process the pathogen activates a cascade of molecular, biochemical, and physiological processes, the result of which is the release of substances detrimental to the host and the development of disease (See, e.g., Scientific American Medicine, W.H. Freeman and Co., San Francisco, 1995; Agrios, G. N., Plant Pathology, Academic Press, 1988). The pathogenic effects of microbes are produced in a variety of ways.
Some pathogens act through secreted products. Diphtheria, for instance, is caused by the bacillus, Cornynebacterium diptheriae. This organism is inhaled by the host and establishes infection in the upper respiratory tract. While the bacterium does not itself invade the bloodstream, its powerful toxins do. These toxins are then absorbed by the cells of the body, enzyme function is impaired, and host cells are destroyed.
Other diseases are the result of the body""s reaction to a pathogen. For example, in pneumonia, a disease caused by Streptococcus pneumoniae, infection causes an outpouring of fluid and cells into the air sacs of the lungs, interfering with respiration. Fungal infections of the skin similarly result from such inflammatory responses.
Yet other bacteria are opportunistic pathogens. Pseudomonas aeruginosa, for example, infects patients with thermal burns and patients who are immunodeficient or otherwise immunologically compromised. P. aeruginosa infections can be acute and localized as in corneal ulcers and otitis media, chronic as in the lungs of cystic fibrosis patients, or systemic following bloodstream invasion.
Plant pathogenic diseases are also of concern because they cause damage to plants and plant products. Phytopathogens produce disease in plants by any number of methods including: (1) consuming host cell nutrients; (2) killing or disrupting host cell metabolism through toxins, enzymes, or growth-regulators; (3) affecting photosynthesis by inducing chlorosis (e.g., by degrading chloroplasts); and (4) blocking conductive tissues and interfering with normal physiological processes.
Crop plants, ornamentals, trees, and shrubs are especially vulnerable to diseases caused by bacteria, fungi, viruses, and nematodes. Phytopathogenic bacteria, for example, cause the development of many disease symptoms including leaf spots and blights, soft-rots, wilts, overgrowths, scabs, and cankers. Bacterial diseases occur most commonly on vegetables (and some ornamentals) that have fleshy storage tissues, such as potatoes, carrots, onions, iris, or hyacinth. They may also occur in plants bearing fleshy fruit (such as cucumber, squash, eggplant, or tomato), as well as in leafy plants (such as cabbage, celery, lettuce, or spinach). Plant bacterial diseases occur throughout the world and cause serious damage to crops in the field, in transit, and in storage.
The mechanisms of plant pathogenesis are many and varied. One bacterial phytopathogen Erwinia, for example, causes plant diseases such as soft-rot and fire-blight by penetrating a plant through a wound or an accessible natural opening. Once inside, the bacteria secrete enzymes which break down the plant""s middle lamellae, resulting in the maceration of tissue and ultimately cell death. Other bacteria, such as certain strains of Pseudomonas, may interfere with water translocation by disrupting xylem within the plant. Pseudomonads invade the xylem of roots and stems and, once inside, secrete enzymes and toxins which destroy the plant. Still other phytopathogenic bacteria, like Agrobacterium and Corynebacterium, stimulate cell division and cell enlargement in affected tissues. This generally leads to the development of amorphous overgrowths, galls, or tumors on roots, stems, or other organs (e.g., crown gall caused by Agrobacterium tumefaciens), or in the proliferation of infected organs (e.g., hairy root caused by Agrobacterium rhizogenes).
Prompt identification of the causative organism is essential to the appropriate selection of anti-pathogenic agents and successful management of clinical and agricultural infections. However, the extensive use of anti-pathogenic agents, such as sulfonamides, tetracyclines, ampicillins, cephalosporins, and aminoglycosides, in both medicine and agriculture has strongly favored the selection of resistant microbial species. This is especially true of bacterial strains containing transmissible resistance plasmids. For example, outbreaks of nosocomial infections from highly resistant strains of Serratia, Klebsiella, Pseudomonas, Acinetobacter, Enterobacter, and Streptococcus have become important and recurrent problems. As a result of selecting resistant strains, over the past few decades, P. aeruginosa has emerged as an important and problematic clinical pathogen, causing between 10% and 20% of infections in hospitals. Currently, several aminoglycosides and third-generation cephalosporins are efficacious against P. aeruginosa, but the relative ease with which P. aeruginosa acquires resistance necessitates the search for new compounds as potential replacements or alternative therapies.
We have discovered that common pathogenic virulence factors are involved in the infection and pathogenicity of both animal and plant hosts. The identification of such host-independent virulence factors has facilitated improved screening methods designed to evaluate and identify therapeutic agents useful for inhibiting pathogenesis in either animal or plant hosts, or both. Furthermore, our discovery provides the basis for screening methods useful for identifying a variety of new virulence factors. Identification of such virulence factors also facilitates the development of targeted reagents for use as anti-pathogens.
In a first aspect, therefore, the invention generally features a method for identifying a compound which is capable of inhibiting a pathogen in a eukaryotic host organism. The method involves (a) exposing (either sequentially or simultaneously) at least two different eukaryotic host organisms, at least one of the organisms being a non-rodent, to a single pathogen in the presence of at least one candidate compound; and (b) identifying a compound that inhibits the pathogen in each of the eukaryotic host organisms.
In preferred embodiments, the pathogen is a bacterium (e.g., Pseudomonas aeruginosa UCBPP-PA14); the eukaryotic host organisms include a vertebrate (e.g., a non-rodent) and a plant, a vertebrate and an invertebrate; or an invertebrate and a plant. Preferably, the invertebrate is a nematode (e.g., a member of the genus Caenorhabditis); and the plant is a crucifer (e.g., a member of the genus Arabidopsis). In other preferred embodiments, each of the eukaryotic host organisms is a plant; is a vertebrate; or is an invertebrate.
In a second aspect, the invention generally features a method for identifying a compound which is capable of inhibiting a pathogen in a non-rodent eukaryotic host organism. The method involves (a) exposing a non-rodent eukaryotic host organism to a single pathogen in the presence of at least one candidate compound; and (b) identifying a compound that inhibits the pathogen in the eukaryotic host organisms.
In one preferred embodiment, the pathogen is a bacterium (e.g., Pseudomonas aeruginosa UCBPP-PA14), and the non-rodent eukaryotic host organism is a nematode (e.g., a member of the genus Caenorhabditis), and the plant is a crucifer (e.g., is a member of the genus Arabidopsis). In a second preferred embodiment, the pathogen is a bacterium (e.g., Pseudomonas aeruginosa UCBPP-PA14), and the non-rodent eukaryotic host organism is a plant (e.g., is a member of the genus Arabidopsis).
In a third aspect, the invention generally features a method for identifying a pathogenic virulence factor. The method involves (a) identifying a pathogen which is capable of infecting at least two different eukaryotic host organisms, at least one of the organisms being a non-rodent; (b) generating a mutant of the pathogen; (c) exposing (either sequentially or simultaneously) each of the organisms to the mutated pathogen; (d) determining whether the mutated pathogen is capable of causing disease in each of the organisms, a reduction of disease in both of the organisms relative to that caused by the wild-type pathogen indicating a mutation in a pathogenic virulence factor; and (e) using the mutation as a marker for identifying the pathogenic virulence factor.
In a fourth aspect, the invention generally features a method for mutating a pathogenic virulence factor. The method involves: (a) identifying a pathogen which is capable of infecting at least two different eukaryotic host organisms, at least one of the organisms being a non-rodent; (b) generating a mutant of the pathogen; (c) exposing (either sequentially or simultaneously) each of the organisms to the mutated pathogen; and (d) determining whether the mutated pathogen is capable of causing disease in each of the organisms, a reduction of disease in both of the organisms relative to that caused by the wild-type pathogen indicating a mutation in a pathogenic virulence factor.
In a fifth aspect, the invention generally features a method of reducing the virulence of a pathogen. The method involves (a) identifying a pathogen which is capable of infecting at least two different eukaryotic host organisms, at least one of the organisms being a non-rodent; (b) generating a mutant of the pathogen; (c) exposing (either sequentially or simultaneously) each of the organisms to the mutated pathogen; and (d) determining whether the mutated pathogen is capable of causing disease in each of the organisms, a reduction of disease in both of the organisms relative to that caused by the wild-type pathogen indicating a reduction in pathogen virulence.
By xe2x80x9cinhibiting a pathogenxe2x80x9d is meant the ability of a candidate compound to decrease, suppress, attenuate, diminish, or arrest the development or progression of a pathogen-mediated disease or an infection in a eukaryotic host organism. Preferably, such inhibition decreases pathogenicity by at least 5%, more preferably by at least 25%, and most preferably by at least 50%, as compared to symptoms in the absence of candidate compound in any appropriate pathogenicity assay (for example, those assays described herein). In one particular example, inhibition may be measured by monitoring pathogenic symptoms in a host organism exposed to a test compound or extract, a decrease in the level of symptoms relative to the level of pathogenic symptoms in a host organism not exposed to the compound indicating compound-mediated inhibition of the pathogen.
By xe2x80x9cnon-rodentxe2x80x9d is meant any organism that is not a mouse, a rat, a guinea pig, or a hamster.
By xe2x80x9cpathogenic virulence factorxe2x80x9d is meant a cellular component (e.g., a protein such as a transcription factor) without which the pathogen is incapable of causing disease or infection in a eukaryotic host organism.
The invention provides long awaited advantages over a wide variety of standard screening methods used for distinguishing and evaluating the efficacy of a compound against microbial pathogens. For example, the screening methods described herein allow for the simultaneous evaluation of host toxicity as well as anti-pathogen potency in a simple in vivo screen. Moreover, the methods of the invention allow one to evaluate the ability of a compound to inhibit microbial pathogenesis, and, at the same time, to evaluate the ability of the compound to stimulate and strengthen a host""s response to pathogenic attack.
Accordingly, the methods of the invention provide a facile means to identify compounds that are safe for use in eukaryotic host organisms (i.e., compounds which do not adversely affect the normal development and physiology of the organism), and efficacious against pathogenic microbes (i.e., by suppressing the virulence of a pathogen). In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for anti-pathogenic effect with high-volume throughput, high sensitivity, and low complexity. The methods are also relatively inexpensive to perform and enable the analysis of small quantities of active substances found in either purified or crude extract form. Furthermore, the methods disclosed herein provide a means for identifying anti-pathogenic compounds which have the capability of crossing eukaryotic cell membranes and which maintain therapeutic efficacy in an in vivo method of administration.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.